Lo, Cecilia IBN9631544 Gap junctions are membrane pores that connect adjacent cells. These molecular pores allow certain kinds of amall organic molecules and ions to pass between cells and thus represent a form of intercellular communication. As gap junctions are found in almost all cells and tissues, and usually are expressed from very early stages of embryogenesis, it is likely that they may play an important role in regulating tissue homeostasis and development. The most definitive evidence of a role for gap junctions in development has surfaced with the recent success of Kidder and Rossant and their colleagues in making a Cx43 gene knock out (KO) mouse line. Cx43 is one member of the gap junction gene family that is widely expressed in a variety of tissues, both in the embryo and in the adult. Mice homozygote for the Cx43 KO allele die soon after birth from heart conotruncal or heart outflow tract defects. As occipital neural crest cells are known to play an important role in morphogenesis of the outflow tract, the phenotype of the KO mice may indicate a role for Cx43 gap junctions in neural crest development. This is further suggested by the finding of conotruncal heart defects and other defects associated with structures of neural crest origin in transgenic mice containing gain or loss of function Cx43 constructs Based on these observations, the investigators hypothesize that Cx43 gap junctions may play an important role in regulating the emergence and migration of neural crest cells, in particular neural crest cells that participate in heart morphogenesis. They plan to carry out experiments outlined in four specific aims. These studies will focus on the Cx43 KO mice, examining how the complete absence of Cx43 may affect the migration and gap junctional communication (GJC) competency of neural crest cells. Analysis will be carried out with neural crest cells cultured in vitro, and also in the living embryo. This combination of in vitro and in vivo studies should reveal if the migrat ory behavior of neural crest cells may be affected by the modulation of gap junctional communication resulting from the loss of Cx43 function. The outcome of these studies will serve as the basis for the planning of future experiments for further examining the mechanism by which perturbations in Cx43 function may lead to conotruncal heart defects, i.e. how is the proliferation, survival, and differentiation of cardiac neural crest cells affected by changes in GJC, and also what might be the signaling pathways disrupted by these changes in GJC. Accordingly, the investigators: 1) Characterize the migration competency of neural crest cells in vitro by monitoring neural crest outgrowth in neural tube explant cultures. 2) Characterize neural crest cell emigration in vivo using DiI method of cell labeling. 3) Quantitate the level of gap junctional communication in neural crest cells with dye injection studies of neural tube explant cultures. and 4) Characterize gap junctional communication in living embryos, specifically in the region containing neural crest cells destined for the heart outflow tract.
